The Daktari diagnostic platform-based on microfluidic sample processing and electrochemical detection-is designed for use by a nurse or primary health care worker with minimal training; total hands-on time is 60 seconds. The initial Daktari CD4 Assay has been developed for performing CD4 cell counts from a 10 L fingerstick blood sample in less than ten minutes, at low cost. Under this project, we will develop an HCV Viral Load Assay on the Daktari platform. The HCV Viral Load Assay will incorporate blood collection, sample processing, HCV capture, and electrical detection in an integrated, disposable plastic cartridge. In Phase I, we will establish the feasibility of whole HCV virus magnetic capture and lysis, and electrical detection of released viral protein. First, the binding specificity and sensiivity of a selected panel of HCV capture agents will be evaluated in both plasma and whole blood. In parallel, we will optimize and evaluate the threshold of detection of an electrical detection method based on a sandwich immunoassay, to detect released HCV core antigen using a gold-nanoparticle/silver deposition signal amplification cascade. The electrical detection method will make use of Daktari's novel electrode design. Should the data from these two studies determine technical feasibility, Phase II of the project will integrate blood collection, HCV capture and lyss, and electrical detection into a Daktari microfluidic cartridge, yielding a complete HCV Viral Load Assay ready for commercial scale-up, for use in Daktari's handheld instrument. First, the HCV capture protocol, consisting of magnetic mixing and concentration using microparticles functionalized with the anti-HCV capture agent identified in Phase I, will be optimized and adapted to the Daktari cartridge production format. Second, the electrical detection module identified in Phase I will be integrated, and the layout of the fluidic paths (including reagent storage and handling) will be designed and tested, ultimately leading to a complete HCV Viral Load Assay cartridge. Finally, the current Daktari instrument will be modified to incorporate the permanent magnets required for magnetic capture of HCV virions, and an additional mechanical actuator for reagent transport. The final deliverable will be a simple, low-cost instrument capable of being used at the primary care level, to quantify circulating HCV and identify patients with active HCV infection in need of treatment.